Sustained release poloxamer containing pharmaceutical compositions

ABSTRACT

A thermo-reversible thermoplastic pharmaceutical composition, comprising a botulinum toxin and a biocompatible poloxamer which provides thermoreversibility to the composition and additionally stabilizes the botulinum toxin, is described. The pharmaceutical composition can be administered to a patient as a liquid, and gels after administration into a sustained release drug delivery system from which the biologically active botulinum toxin is released over a multi-day period thereby localizing the drug as a depot and controlling release to enhance the therapeutic effect per dose.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 12/036,139,filed Feb. 22, 2008, which is hereby incorporated herein by reference inits entirety.

BACKGROUND

The present invention relates to thermosensitive, thermo-reversiblepharmaceutical compositions. In particular, the present inventionrelates to sustained release, gelable (thermosensitive) botulinum toxinpharmaceutical compositions formulated with a poloxamer.

A pharmaceutical composition is a formulation which contains at leastone active ingredient (such as a botulinum toxin) as well as, forexample, one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents, and is suitable for administrationto a patient to achieve a desired diagnostic result or therapeuticeffect. The pharmaceutical compositions disclosed herein havediagnostic, therapeutic, cosmetic and/or research utility.

For storage stability and convenience of handling, a pharmaceuticalcomposition can be formulated as a lyophilized (i.e. freeze dried) orvacuum dried powder which can be reconstituted with a suitable fluid,such as saline or water, prior to administration to a patient.Alternately, the pharmaceutical composition can be formulated as a readyto use aqueous solution or suspension. A pharmaceutical composition cancontain a proteinaceous active ingredient. Unfortunately, a proteinactive ingredient can be very difficult to stabilize (i.e. maintained ina state where loss of biological activity is minimized), resultingtherefore in a loss of protein and/or loss of protein activity duringthe formulation, reconstitution (if required) and during the period ofstorage prior to use of a protein containing pharmaceutical composition.Stability problems can occur because of protein denaturation,degradation, dimerization, and/or polymerization. Various excipients,such as albumin and gelatin have been used with differing degrees ofsuccess to try and stabilize a protein active ingredient present in apharmaceutical composition. Additionally, cryoprotectants such asalcohols have been used to reduce protein denaturation under thefreezing conditions of lyophilization.

Thermosensitive pharmaceutical compositions, which form in-situ gels,are known. See eg U.S. Pat. No. 5,278,201. Poloxamers are nontoxic blockcopolymers of poly(ethylene oxide), poly(propylene oxide) andpoly(ethylene oxide) (PEO-PPO-PEO). Certain poloxamers exhibitreversible thermal gelation. Thus a solution of a protein and apoloxamer prepared at low temperatures and injected will form a gel asit warms to body temperature. Subsequently the protein is slowlyreleased from the gel. A gelable, thermo-reversible formulationcomprising poloxamer 407 at a 22 wt % concentration has been preparedwith the model protein drugs α-chymotrypsin and lactate dehydrogenase.Stratton L., et al., Drug delivery matrix containing native proteinprecipitates suspended in a poloxamer gel, J Pharm Sci 86(9); 1006-1010,September 1996. Formulations of certain adhesive proteins and poloxamer127 have been made. Huang K., et al., Synthesis and characterization ofself assembling block copolymers containing adhesive moieties, PolymerPreprints 2001, 42(2), 147-148. Additionally, poloxamer 188 andpoloxamer 407 have been used an excipients in protein drugpharmaceutical compositions. Jeong B., et al., Thermosensitive sol-gelreversible hydrogels, Adv Drug Del Rev, 54(1); 37-51, Jan. 17, 2002.Published patent application WO 2007/041664 discloses use apharmaceutical composition comprising a botulinum toxin and a poloxamer188.

Botulinum toxins have been used for various therapeutic and cosmeticpurposes including treating cervical dystonia, blepharospasm,strabismus, spasticity, headache, hyperhidrosis, overactive bladder,rhinitis, bruxism, enlarged prostate, achalasia, anismus, sphincter ofOddi malfunction, acne, tremors, juvenile cerebral palsy, and facialwrinkles.

Commercially available botulinum toxin containing pharmaceuticalcompositions include BOTOX® (Botulinum toxin type A neurotoxin complexwith human serum albumin and sodium chloride) available from Allergan,Inc., of Irvine, Calif. in 100 unit vials as a lyophilized powder to bereconstituted with 0.9% sodium chloride before use), DYSPORT®(Clostridium botulinum type A toxin haemagglutinin complex with humanserum albumin and lactose in the formulation), available from IpsenLimited, Berkshire, U.K. as a powder to be reconstituted with 0.9%sodium chloride before use), and MYOBLOC™ (an injectable solutioncomprising botulinum toxin type B, human serum albumin, sodiumsuccinate, and sodium chloride at about pH 5.6, available from SolsticeNeurosciences, Inc., South San Francisco, Calif.).

Botulinum toxin is a large protein for incorporation into apharmaceutical formulation (the molecular weight of the botulinum toxintype A complex is 900 kD) and is inherently fragile and labile. The sizeof the toxin complex makes it much more friable and labile than smaller,less complex proteins, thereby compounding the formulation and handlingdifficulties if botulinum toxin stability is to be maintained. Hence, abotulinum toxin stabilizer must be able to interact with the toxin in amanner which does not denature, fragment or otherwise detoxify the toxinmolecule or cause disassociation of the non-toxin proteins present inthe toxin complex.

As the most lethal known biological product, exceptional safety,precision, and accuracy are called for at all steps of the formulationof a botulinum toxin containing pharmaceutical composition. Thus, abotulinum toxin stabilizer should not itself be toxic or difficult tohandle so as to not exacerbate the already extremely stringent botulinumtoxin containing pharmaceutical composition formulation requirements.

Since botulinum toxin was the first microbial toxin to be approved (bythe U.S. Food and Drug Administration in 1989) for injection for thetreatment of human disease, specific protocols had to be developed andapproved for the culturing, bulk production, formulation into apharmaceutical and use of botulinum toxin. Important considerations aretoxin purity and dose for injection. The production by culturing and thepurification must be carried out so that the toxin is not exposed to anysubstance that might contaminate the final product in even trace amountsand cause undue reactions in the patient. These restrictions requireculturing in simplified medium without the use of animal meat productsand purification by procedures not involving synthetic solvents orresins. Preparation of toxin using enzymes, various exchangers, such asthose present in chromatography columns and synthetic solvents, canintroduce contaminants and are therefore excluded from preferredformulation steps. Furthermore, botulinum toxin type A is readilydenatured at temperatures above 40 degrees Centigrade, loses toxicitywhen bubbles form at the air/liquid interface and denatures in thepresence of nitrogen or carbon dioxide.

Particular difficulties exist to stabilize botulinum toxin type A,because type A consists of a toxin molecule of about 150 kD innoncovalent association with nontoxin proteins weighing about 750 kD.The nontoxin proteins are believed to preserve or help stabilize thesecondary and tertiary structures upon which toxicity is dependant.Procedures or protocols applicable to the stabilization of nonproteinsor to relatively smaller proteins are not applicable to the problemsinherent with stabilization of the botulinum toxin complexes, such asthe 900 kD botulinum toxin type A complex. Thus while from pH 3.5 to 6.8the type A toxin and non toxin proteins are bound togethernoncovalently, under slightly alkaline conditions (pH>7.1) the verylabile about 150 kD neurotoxic component of a botulinum toxin isreleased from the botulinum toxin complex. Xeomin™ is the trade name fora neurotoxic component botulinum toxin type A pharmaceutical compositionavailable from Merz Pharmaceuticals (Frankfurt, Germany).

In some instances botulinum toxins, when used as therapeutic drugs, areknown to migrate from the site of injection at various rates anddistances, sometimes resulting in loss of effect at the desired muscle.

Solid botulinum toxin implants are known. See e.g., U.S. Pat. Nos.6,306,423; 6,312,708, for a discussion of exemplary solid implants andapplications. Additionally formulation of a botulinum toxin in a viscouscarrier such as a hyaluronic acid is known; U.S. applications Ser. Nos.11/954,629, and 11/954,602, filed Dec. 12, 2007.

What is needed is a biocompatible, gelable (thermoplastic)pharmaceutical composition comprising a stabilized botulinum toxin sothat the composition can be administered as a liquid yet forms asustained release gel upon administration; thereby localizing the effectand controlling release of the toxin to enhance the effect per dose.

SUMMARY

The present invention fulfills this need and provides a gelable,thermoreversible, thermoplastic botulinum toxin pharmaceuticalcomposition that can be administered as a liquid and form a gel fromwhich the botulinum toxin exhibits a sustained release uponadministration of the pharmaceutical composition. Additionally, thepresent invention provides the additional advantage in that the compoundwhich provides the thermo-reversible characteristic to the compositioncan also stabilize the botulinum toxin present in the pharmaceuticalcomposition.

In one embodiment the present invention provides a thermoplasticthermoreversible, botulinum toxin pharmaceutical composition formulatedwith a poloxamer. Importantly, besides providing the thermoreversible,thermoplastic characteristics of the pharmaceutical composition thepoloxamer can also act to stabilize the botulinum toxin.

DEFINITIONS

As used herein the words or terms set forth below have the followingmeaning.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration”, or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject. Thepharmaceutical compositions disclosed herein are “locally administered”by e.g. intramuscular (i.m.), intradermal, subcutaneous administration,intrathecal administration, intraperitoneal (i.p.) administration,topical (transdermal) and implantation (i.e. of a slow-release device)routes of administration.

“Botulinum toxin” means: (1) a neurotoxin produced by Clostridiumbotulinum, as well as a botulinum toxin (or the light chain or the heavychain thereof) made recombinantly by a non-Clostridial species; (2) thebotulinum toxin serotypes A, B, C, D, E, F and G; (3) a botulinum toxincomplex (i.e. the 300, 600 and 900 kDa complexes) as well as theneurotoxic component of a purified botulinum toxin (i.e. about 150 kDa),or; (4) a modified botulinum toxin, a pegylated (with a PEG), chimeric,recombinant, hybrid, wild-type botulinum toxins, botulinum toxinconstructs, endopeptidases, chemically-modified botulinum toxins(pegylated botulinum toxin), and retargeted botulinum toxin, whichretains the intracellular ability to inhibit acetylcholine release froma cell.

“Entirely free” (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Pharmaceutical composition” means a formulation in which an activeingredient can be a Clostridial neurotoxin, such as a botulinum toxin.The word “formulation” means that there is at least one additionalingredient in the pharmaceutical composition besides a Clostridialneurotoxin active ingredient. A pharmaceutical composition is thereforea formulation which is suitable for diagnostic, therapeutic or cosmeticuse (i.e. by intramuscular or subcutaneous injection or by insertion ofa depot or implant or topical application) to a subject, such as a humanpatient. The pharmaceutical composition can be in a lyophilized orvacuum dried condition; a solution formed after reconstitution of thelyophilized or vacuum dried pharmaceutical composition with saline orwater, or; as a solution which does not require reconstitution. Asstated, a pharmaceutical composition can be liquid or solid, for examplevacuum-dried. The constituent ingredients of a pharmaceuticalcomposition can be included in a single composition (that is all theconstituent ingredients, except for any required reconstitution fluid,are present at the time of initial compounding of the pharmaceuticalcomposition) or as a two-component system, for example a vacuum-driedcomposition reconstituted with a diluent such as saline which diluentcontains an ingredient not present in the initial compounding of thepharmaceutical composition. A two-component system provides the benefitof allowing incorporation of ingredients which are not sufficientlycompatible for long-term shelf storage with the first component of thetwo component system. For example, the reconstitution vehicle or diluentmay include a preservative which provides sufficient protection againstmicrobial growth for the use period, for example one-week ofrefrigerated storage, but is not present during the two-year freezerstorage period during which time it might degrade the toxin. Otheringredients, which may not be compatible with a Clostridial toxin orother ingredients for long periods of time, may be incorporated in thismanner; that is, added in a second vehicle (i.e. in the reconstitutionfluid) at the approximate time of use.

“Sustained release” means that the therapeutic agent (i.e. a botulinumtoxin) contained by a pharmaceutical composition (such as apharmaceutical composition comprising a poloxamer) is released from thepharmaceutical composition over a period of time between about 5 daysand about 1 year.

“Stabilizer” (or “primary stabilizer”) is a compound that assists topreserve or maintain the biological structure (i.e. the threedimensional conformation) and/or biological activity of a protein (suchas a botulinum toxin). More than one stabilizer can be included in apharmaceutical composition. Examples of stabilizers are surfactants,polymers, polyols, a poloxamer, albumin, gelatin, trehalose, proteins,sugars, polyvinylpyrrolidone, N-acetyl-tryptophan (“NAT”)), caprylate(i.e. sodium caprylate), a polysorbate (i.e. P80), amino acids, anddivalent metal cations such as zinc. A pharmaceutical composition canalso include a preservative such as a benzyl alcohol, cresols, benzoicacid, phenol, parabens and sorbic acid.

“Stabilizing”, “stabilizes”, or “stabilization” mean that an activepharmaceutical ingredient (“API”) retains at least 20% and up to 100% ofits biological activity (which can be assessed as potency or as toxicityby an in vivo LD₅₀ or ED₅₀ measure) in the presence of a compound whichis stabilizing, stabilizes or which provides stabilization to the API.For example, upon (1) preparation of serial dilutions from a bulk orstock solution, or (2) upon reconstitution with saline or water of alyophilized, or vacuum dried botulinum toxin containing pharmaceuticalcomposition which has been stored at or below about −2 degrees C. forbetween six months and four years, or (3) for an aqueous solutionbotulinum toxin containing pharmaceutical composition which has beenstored at between about 2 degrees and about 8 degrees C. for from sixmonths to four years, the botulinum toxin present in the reconstitutedor aqueous solution pharmaceutical composition has (in the presence of acompound which is stabilizing, stabilizes or which providesstabilization to the API) greater than about 20% and up to about 100% ofthe potency or toxicity that the biologically active botulinum toxin hadprior to being incorporated into the pharmaceutical composition.

“Substantially free” means present at a level of less than one percentby weight of the pharmaceutical composition.

“Therapeutic agent” means an active pharmaceutical ingredient (API)which can have therapeutic, cosmetic, and/or research use or benefitwhen administered to a patient. The therapeutic agent can be for examplea steroid, antibiotic, or protein.

“Thermoplastic” is synonymous with “thermosensitive” and means acompound or composition which is a liquid or a low viscosity solution(i.e. viscosity less that 500 cps at 25° C. at a shear rate of about0.1/second) at a low temperature (between about 0° C. to about 10° C.),but which is a higher viscosity ((i.e. viscosity less that 10,000 cps at25° C. at a shear rate of about 0.1/second) gel at a higher temperature(between about 30° C. to about 40° C. such as at about 37° C.)

In particular embodiments, a thermoplastic, thermoreversible,pharmaceutical composition is disclosed comprising a biologically activebotulinum toxin, and a thermoplastic poloxamer, where the poloxamerstabilizes the botulinum toxin so that the botulinum toxin retainsbiological activity upon release of the botulinum toxin from thepharmaceutical composition in vivo. The botulinum toxin is selected fromthe group consisting of the botulinum toxins types A, B, C₁, D, E, F andG and is preferably a botulinum toxin type A, known for its long lastingeffect. An exemplary thermo-reversible poloxamer is a poloxamer 407, anexample of which can be obtained from the BASF Corporation, Parsippany,N.J., under the name F-127.

In some embodiments, the poloxamer can be present at a concentration offrom about 15 wt % to about 25 wt % of the pharmaceutical composition.

In one specific embodiment, the thermoreversible, thermoplastic,pharmaceutical composition comprises a biologically active botulinumtoxin type A and a thermoplastic poloxamer 407 present at aconcentration of about 15 wt % to about 25 wt % of the pharmaceuticalcomposition, which stabilizes the botulinum toxin so that the botulinumtoxin retains biological activity upon release of the botulinum toxinfrom the pharmaceutical composition in vivo.

A method for treating a medical or cosmetic condition is also disclosedherein, the method comprising the step of administering to a patient athermoplastic, thermo-reversible pharmaceutical composition comprising abiologically active botulinum toxin and a thermoplastic,thermoreversible, poloxamer such that the poloxamer stabilizes thebotulinum toxin so that the botulinum toxin retains biological activityupon release of the botulinum toxin from the pharmaceutical compositionin vivo and the pharmaceutical composition is administered as a liquidand becomes a gel after the administration, to provide therapeuticamounts of the botulinum toxin released from the composition in vivo forat least 1 week after the administration. The thermoreversible,thermoplastic is a poloxamer 407 and is present at a concentration ofabout 15 wt % to about 25 wt % of the pharmaceutical composition. Insome methods, a step of placing a cooling or heating element (such as,for example, a hot or cold pad, bottle, packet of ice or warm water andthe like) is placed over an area of administration, before or after theadministration step, in order to warm or cool the area to decrease orincrease the viscosity of the administered thermoplastic,thermoreversible, pharmaceutical composition in situ and after itsadministration to the patient.

Exemplary medical conditions that can be so treated include glabellarlines, crows feet, marionette lines, nasolabial lines, horizontal linesof the forehead or any combination thereof. Additional exemplary medicalor cosmetic conditions include treating at least one of overactivebladder, hyperhidrosis, benign prostatic hyperplasia and a dystonia,where the botulinum toxin is selected from the group consisting of thebotulinum toxins types A, B, C₁, D, E, F and G. As above, the preferredbotulinum toxin is botulinum toxin type A.

In particular embodiments, the botulinum toxin type A is present in thethermoreversible, thermoplastic, pharmaceutical composition in an amountfrom about 5 units to about 2750 units. The total amount (units) of abotulinum toxin to be administered to a patient is determined by theattending medical professional.

Also herein disclosed is a process for making a thermoreversible,thermoplastic, gelable, pharmaceutical composition, comprising the stepsof dissolving a thermoplastic poloxamer in a solvent at a temperaturebelow about 37 degrees Centigrade, adding and mixing a botulinum toxinto the thermoplastic poloxamer in the solvent to thoroughly disperse thebotulinum toxin therein. The poloxamer can be present in particularembodiments at a concentration from about 15 wt % to about 25 wt % ofthe pharmaceutical composition, thereby making the thermoplastic,gelable, pharmaceutical composition.

In a particular process for making the thermoreversible, thermoplastic,gelable pharmaceutical composition, a botulinum toxin type A or B isutilized. An exemplary amount added can be from about 1 to about 2000units of the botulinum toxin type A or from about 50 to about 25,000units of a botulinum toxin type B, to be administered to a patient inneed thereof. The process can also include adding an additive, forpreservative or stabilizing purposes, for example, to form the finalthermoplastic, gelable, pharmaceutical composition. In specificexamples, the solvent utilized in the method of making thethermoplastic, gelable, pharmaceutical composition is water or a salinesolution and the process of making the thermoplastic, gelable,pharmaceutical composition is carried out within a cold room having antemperature of below about 37 degrees Centigrade, or between about 0 toabout 8 degrees Centigrade, for example.

Also disclosed are methods for treating a medical or cosmetic condition,that comprise the steps of administering to a patient athermoreversible, thermoplastic, pharmaceutical composition thatincludes a biologically active botulinum toxin and a thermoreversible,thermoplastic poloxamer, wherein the poloxamer stabilizes the botulinumtoxin and is a gel at room temperature (e.g the temperature of anenclosed space at which human beings are usually accustomed, e.g. fromabout 17° C. to about 25° C. Before administration, the pharmaceuticalcomposition is cooled below the room temperature to reduce its viscosity(liquefy) the pharmaceutical composition and is thereafter drawn into asyringe and injected into the patient, where the thermoplasticpharmaceutical composition gels to deliver therapeutic amounts of thebotulinum toxin are released from the composition in vivo for at least 1week after administration. In particular embodiments, the thermoplasticpoloxamer is a poloxamer 407 and is present at a concentration of about15 wt % to about 25 wt % of the pharmaceutical composition.

The thermoplastic, pharmaceutical composition is thermo-reversible, thatis, its viscosity can be increased and/or decreased based ontemperature, and is reversible. For example, the thermoreversible,thermoplastic poloxamer such as poloxamer 407 at about 20% wt (and hencethe thermoplastic, pharmaceutical composition) can have a firstviscosity at a first temperature (e.g. from about 0 centipoise (cP) atabout 0 to about 16 degrees Centigrade), have its temperature raised toincrease its viscosity to a second viscosity that is higher relative tothe first viscosity (e.g. from about 50 cP to about 6000 cP at about 18to about 22 degrees Centigrade), and then is reversible, e.g. loweringits temperature, decreasing its viscosity relative to the secondviscosity, for example. A change in weight % of poloxamer 407 in acomposition will alter its viscosity/temperature profile.

Each and every feature described herein, and each and every combinationof two or more of such features, is included within the scope of thepresent invention, provided that the features included in such acombination are not mutually inconsistent.

DESCRIPTION

I have discovered new thermo-reversible depot pharmaceuticalcompositions (formulations) for a botulinum toxin. My invention is basedon the discovery that certain thermo-reversible poloxamers showremarkable compatibility with botulinum toxins. These depot systems canbe thermally manipulated (pre & post injection) to control migration anddistribution of the botulinum toxin. No combinations of botulinum toxinsand poloxamer 407 are known.

A formulation within the scope of my invention is administered as aliquid and the polymer gels (cures) in situ at the site of localadministration.

An embodiment of my invention can comprise a triblock PEO-PPO-PEOcopolymer compounds, also known as pluronics or poloxamers, which atcertain concentrations can form thermo-reversible gels which can beadministered (as by injection) as a low viscosity liquid that rapidlyincreases in viscosity after injection. The resulting high viscositymatrix is adhesive, biodegradable and biocompatible and uponadministration forms a depot from which the botulinum toxin can bereleased, thereby providing a sustained or extended release drugdelivery system. In this manner a lower dose of the botulinum toxin canbe used. Such a pharmaceutical composition can be administered pre-mixedor as a simple reconstitution vehicle or its several compartmentscombined at the time of administration, as by use of a dual chambersyringe.

I have found that botulinum neurotoxins are very stable in poloxamers,such as poloxamer 407, for example. This is surprising because of thecomplex structural nature of these toxins. For example, three separateprotein domains (binding, translocation, enzymatic) must be conserved inorder to maintain biological activity of the naked 150 kD toxin.Surfactants are chaotropic and therefore generally disrupt proteinconformations. It is therefore surprising to find compatibility betweenthese molecules and surfactants. The 900 kD toxins are protein complexeswith neurotoxin associated proteins (NAPs), which stabilize the 150 kDportion. Surfactants would be expected to disrupt the protein complex,thereby destabilizing the complex and/or denaturing the 150 kD toxinportion.

The thermo-reversible poloxamer used in the present invention canapparently impart stability to a neurotoxin active ingredient, such as abotulinum toxin, present in the pharmaceutical composition by: (1)reducing adhesion (commonly referred to as “stickiness”) of thebotulinum toxin to surfaces, including the surfaces of laboratoryglassware, vessels, the vial in which the pharmaceutical composition isreconstituted and the inside surface of the syringe used to inject thepharmaceutical composition. Adhesion of the botulinum toxin to surfacescan lead to loss of botulinum toxin and to denaturation of retainedbotulinum toxin, both of which reduce the toxicity of the botulinumtoxin present in the pharmaceutical composition; (2) reducing thedenaturation of the botulinum toxin and/or dissociation of the botulinumtoxin from other non-toxin proteins present in the botulinum toxincomplex, which denaturation and/or dissociation activities can occurbecause of the low dilution of the botulinum toxin present in thepharmaceutical composition (i.e. prior to lyophilization or vacuumdrying) and in the reconstituted pharmaceutical composition; (3)reducing loss of botulinum toxin (i.e. due to denaturation ordissociation from non-toxin proteins in the complex) during theconsiderable pH and concentration changes which take place duringpreparation, processing and reconstitution of the pharmaceuticalcomposition; (4) immobilizing the toxin in a high-viscosity vehicle; and(5) protecting the toxin from deleterious effects of elevatedphysiologic temperature (about 37° C.) and pH by providing a beneficialmicro-environment.

The five types of botulinum toxin stabilizations provided by thepoloxamers disclosed herein conserve and preserve the botulinum toxinand with it native toxicity of the toxin present in the pharmaceuticalcomposition.

My invention also encompasses addition of a preservative, either in thediluent or formulation itself, to allow extended storage. A preferredpreservative is preserved saline containing benzyl alcohol.

The thermo-reversible pharmaceutical compositions of the invention canbe administered using conventional modes of administration. Inparticular embodiments of the invention, the compositions areadministered intradermally, intramuscularly or subcutaneously to thepatient. In addition, the compositions of the invention may beadministered with one or more analgesic or anesthetic agents.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the type, severity, andcourse of the condition being treated, the patient's health and responseto treatment, and the judgment of the treating physician. Accordingly,the methods and dosages of the compositions can be tailored to theindividual patient.

Compositions containing other serotypes of botulinum toxin may containdifferent doses (unit amounts) of the botulinum toxin. For example,botulinum toxin type B can be provided in a composition at a greaterdose (about 50×) than a composition containing botulinum toxin type A.In one embodiment of the invention, botulinum toxin type B may beadministered in an amount between about 1 U/kg and 150 U/kg. Botulinumtoxin type B may also be administered in amounts of up to 20,000 U(mouse units, as described above). In another embodiment of theinvention, botulinum toxin types E or F may be administered atconcentrations between about 0.1 U/kg and 150 U/kg. In addition, incompositions containing more than one type of botulinum toxin, each typeof botulinum toxin can be provided in a relatively smaller dose than thedose typically used for a single botulinum toxin serotype.

EXAMPLES

The following examples set forth specific embodiments of the presentinvention and are not intended to limit the scope of the invention.

In the Examples below the well known mouse lethal dose₅₀ assay (the“MLD50 assay”) was used to determine the potency of the botulinum toxinreleased from the poloxamer formulations made. Depending on thecircumstances, “potency” can mean the recovered potency of the botulinumtoxin or the potency of the botulinum toxin prior to lyophilization.Recovered potency is synonymous with reconstitution potency, recoverypotency and with potency upon reconstitution. The MLD50 assay provides adetermination of the potency of a botulinum toxin in terms of its mouse50% lethal dose or “LD50”. Thus, one unit (U) of a botulinum toxin isdefined as the amount of botulinum toxin which upon intraperitonealinjection kills 50% (i.e. a LD₅₀) of a group of female Swiss Weber miceweighing 17-22 grams each at the start of the assay. The MLD50 assay isa validated method for measuring the potency of a reconstitutedbotulinum toxin or of a reconstituted botulinum toxin formulation. Eachmouse is held in a supine position with its head tilted down and isinjected intraperitoneally into the lower right abdomen at an angle ofabout 30 degrees using a 25 to 27 gauge ⅜″ to ⅝″ needle with one ofseveral serial dilutions of the botulinum toxin in normal saline. Thedeath rates over the ensuing 72 hours for each dilution are recorded. Aminimum of six dilutions at 1.33 dose intervals are prepared andtypically ten animals are used in each dosage group (60 mice employedtherefore). Two reference standard assays are carried out concurrently(additional 60 mice employed). The dilutions are prepared so that themost concentrated dilution produces a death rate of at least 80% of themice injected, and the least concentration dilution produces a deathrate no greater than 20% of the mice injected. There must be a minimumof four dilutions that fall within the monotone decreasing range of thedeath rates. The monotone decreasing range commences with a death rateof no less than 80%. Within the four or more monotone decreasing rates,the two largest and the two smallest rates must be decreasing (i.e. notequivalent). The dilution at which 50% of the mice die within the threeday post injection observation period is defined as a dilution whichcomprises one unit (1 U) of the botulinum toxin. A refined MLD50 assayhas been developed which uses fewer (five instead of six) dilutions at1.15 dose intervals and fewer mice (six instead of ten) per dilutiontested.

Example 1 Botulinum Toxin-Poloxamer 407 Formulations

Experiments were carried out in which a number of botulinumtoxin-poloxamer formulations were made and assessed. The botulinum toxinused in each of the formulations made was lyophilized BOTOX®. The amountof the botulinum toxin used in each of the formulations made in 100units of botulinum toxin type A (BOTOX®).

The poloxamer used in this example was poloxamer 407 obtained from BASF(Lutrol F-127). In each formulation, the poloxamer 407 was used in anamount that constituted 20 weight percent (wt %) of the finalformulation. Poloxamer 407 is supplied as dry powder and is ahydrophilic non-ionic surfactant and a triblock copolymer consisting oftwo hydrophilic blocks (polyethylene glycol) separated by a hydrophobicblock (poly-propylene glycol). The lengths of the two PEG blocks isabout 101 repeating units, while the length of the propylene glycolblock is about 56 repeating units. Solutions of Poloxamer 407, atappropriate concentrations, are liquid under refrigeration, but gel atroom temperature and above (e.g., is a gel at about 37° C.). Poloxamer407 can therefore be reconstituted or stored as a low-viscosity liquidfor easy passage through a needle but then gels into a depot afterinjection into a mammal as it is subjected to increasing temperature.Poloxamer 407 was chosen for the formulations made in this examplebecause of these desirable physical properties combined with unusualtoxin compatibility.

Each formulation was made by a process which combines the solids (recallthat the Poloxamer 407 is supplied as dry powder) in large centrifugetubes with water or saline (inside a cold room at about 2 to about 8degrees Centigrade) and is mixed with a magnetic stir bar, until fullydissolved.

The solutions are stored at from bout 4 to about 15 degrees Centigradeuntil injected/used. The solutions were then used to reconstitute abotulinum toxin type A (BOTOX®) in vials containing 100 units (U), byintroducing the cold solution into the vials with a syringe.

Samples were then heated to 37° C. until gelled to simulate injectioninto a warm body.

Significantly I determined that the thermo-reversible formulations canbe made with from 15-25 wt % poloxamer 407 (available from BASF asLutrol F-127) without significant attenuation of the desired formulationcharacteristics of (1) thermoplasticity, and (2) sustained release ofbiologically active botulinum toxin from the formulations made.

As set forth below, 26 different thermo-reversible poloxamerformulations were made. Each of the 26 formulation made included 20 wt %poloxamer 407 and 100 units of botulinum toxin type A (BOTOX®):

1. 20% Poloxamer 407 in SWFI (sterile water for injection)

2. 20% Poloxamer 407 in 0.9% sodium chloride

3. 20% Poloxamer 407 in preserved (benzyl alcohol) 0.9% sodium chloride

4. 20% Poloxamer 407 with 5% Poloxamer 188

5. 20% Poloxamer 407 with 3% Tween

6. 20% Poloxamer 407 with 5% sucrose

7. 20% Poloxamer 407 with 5% dextran

8. 20% Poloxamer 407 in 10 mM histidine pH 7

9. 20% Poloxamer 407 in 20 mM citrate buffer pH 6

10. 20% Poloxamer 407 in phosphate buffered saline pH 7

11. 20% Poloxamer 407 with 20% propylene glycol

12. 20% Poloxamer 407 with 10% polyethylene glycol

13. 20% Poloxamer 407 with 20 mM Tris buffer pH 7

14. 20% Poloxamer 407 with 3% isopropyl myristate

15. 20% Poloxamer 407 with 5% povidone

16. 20% Poloxamer 407 with 3% lactose

17. 20% Poloxamer 407 with 3% trehalose

18. 20% Poloxamer 407 with 0.5% human serum albumin

19. 20% Poloxamer 407 with 0.5% human serum albumin 900 ug NaCl

20. 20% Poloxamer 407 with 0.5% recombinant human serum albumin

21. 20% Poloxamer 407 with 0.5% gelatin

22. 20% Poloxamer 407 with 0.5% recombinant gelatin

23. 20% Poloxamer 407 with 0.5% hyaluronic acid

24. 20% Poloxamer 407 with 0.5% collagen

25. 20% Poloxamer 407 with 2% hydroxypropyl methylcellulose

26. 20% Poloxamer 407 with 2% lecithin

To determine that active botulinum toxin was released from each of the26 thermo-reversible poloxamer formulations, light chain activity wasmeasured using a fluorescent SNAP-25 substrate coupled with HPLC.Samples incubated with the substrate produce a cleavage product that isseparated by RP-HPLC and detected via fluorescence. The amount ofcleavage product is proportional to enzymatic activity.

Poloxamer 407 can be further manipulated pre and/or post-injection byapplying heat or cold-packs to desired areas (injected and/ornon-injected) achieve the desired effect. Additional ingredients can beadded to the formulation to modify the attributes (causingincreases/decreases in gelling temperatures, for example). Ingredientsto alter osmolarity and pH (buffers) can be added. Administration can betopical rather than injectable; for example, in a transdermal deliveryscheme, the formulation can contain permeation enhancers, and may becombined with a device such as a patch having additional permeationattributes, such as abrasives or microneedles, for example.Preservatives can also be included in the formulation. Colorants, suchas pharmaceutically acceptable dyes, can be included to better visualizethe material before and after application.

Example 2 Use of a Poloxamer-Botulinum Toxin Pharmaceutical Composition

A 48 year old male is diagnosed with a spastic muscle condition, such ascervical dystonia. Between about 50 to about 500 units of botulinumtoxin type A (such as BOTOX®) combined with formulation 2 (20% Poloxamer407 in 0.9% sodium chloride) in Example 1, and is administered byintramuscular injection. The formulation releases therapeutic amounts ofthe botulinum toxin over a 1 month period. Within 1-7 days the symptomsof the spastic muscle condition are alleviated and alleviation of thesymptoms persists for at least about 6 months.

Example 3 Use of a Poloxamer-Botulinum Toxin Pharmaceutical Composition

A 22 year old female sees her physician to report and treat heruncontrollable and excessive armpit, sweating or as its known in themedical arts, axillary sweating. After gravimetric measurement of hersweat production, she is diagnosed as suffering from hyperhidrosis.

About 100 units of botulinum toxin type A (such as BOTOX®) is combinedwith formulation 12 (20% Poloxamer 407 with 10% polyethylene glycol) inExample 1, and is administered by intradermal injection into theaxillary hyperhidrotic area (as determined by Minor's starch-iodinetest). After injection, an ice pack is placed over the injected area,cooling the area and making the injected thermo-reversiblepoloxamer-botulinum toxin pharmaceutical composition less viscous,allowing the attending physician to massage the injected area, allowinga more even spread of the injected composition. Within 7 days, theexcessive axial sweating is reduced and alleviation is observed forabout 8 months.

Example 4 Use of a Poloxamer-Botulinum Toxin Pharmaceutical Composition

A 38 year old woman reports to her dermatologist that she can no longerwithstand the sight of her glabellar lines (dynamic wrinkles between thebrows caused by the contraction of corrugator and/or procerus muscles)and that they have become a source of great consternation. Thedermatologist determines to treat her with a poloxamer-botulinum toxinpharmaceutical composition.

About 500 units of a botulinum toxin type B is combined with formulation25 (20% Poloxamer 407 with 2% hydroxypropyl methylcellulose) in Example1, and is administered by intramuscular injection directly into thecorrugator and procerus muscles. Areas outside the desired treatmentarea are pre-heated (utilizing a heating pad to gradually warm the areasnot injected, for example, from about 37 to about 43 degrees Centigrade,to elevate the temperature relative to the areas of injection) toprevent drug migration into those regions. Within about 7 days, thepatient reports that the glabellar lines have been reduced and the skinbetween her brows is smoother. The alleviation of the wrinkles lasts forabout 4 months.

Similarly, a botulinum toxin type A (BOTOX®) at about 2 units per 0.1 mLof added formulation can be injected at each of about 5 injection sitesin corrugator and procerus muscles for a total dose of about 10 unitsper 0.5 mL of thermo-reversible poloxamer-botulinum toxin pharmaceuticalcomposition. As an additional step, after or before injection into themuscles, a hot pad can be placed over the injection site to warm thearea. Thus, if a hot pad is so placed, the injected poloxamer-botulinumtoxin pharmaceutical composition can gel faster than if injected at justbody temperature. The pad can be between about 37 degrees and about 43degrees Centigrade, for example. The pad can be placed onto the areainjected or to be injected and warmed up to between about 37 degrees andabout 43 degrees Centigrade, for example.

Example 5 Use of a Poloxamer-Botulinum Toxin Pharmaceutical Composition

A 78 year old man is brought to his urologist, complaining of aninability to withhold his urine for any significant amount of time. Theurologist determines that the patient is incontinent and has anoveractive bladder (OAB) and that his detrusor muscle should be injectedwith a poloxamer-botulinum toxin pharmaceutical composition.

About 250 units of a botulinum toxin type A (BOTOX) is combined withformulation 2 (20% Poloxamer 407 in 0.9% sodium chloride) of Example 1(about 250 units of toxin in about 10 mL of formulation 2). Utilizing aflexible cytoscope and standard bladder wall injection equipment (localanesthetic, lubricants, etc. . . . ), the urologist proceeds to injectthe patient's bladder wall at 10 sites (25 units/site) along the lateralwalls, sparing the trigone and dome. Within about 7 days, the patientreports that he is able to hold his urine for many hours at a time, andthat his voiding volume per visit to the urinal has more than doubled.The patient reports relief from his incontinence for approximately 7months.

A bladder wall can also be injected with any one of the formulations(1-26) in Example 1 containing other botulinum toxin types, such as fromabout 50 to about 15,000 units of a botulinum toxin type B, utilizingfrom about 5 mL to about 30 mL of the formulations in Example 1. Thethermo-reversible poloxamer-botulinum toxin pharmaceutical compositionis injected into the bladder wall in about 5 to about 50 injectionsites, as determined by an attending physician, and can include orexclude the trigone, if desired.

Example 6 Use of a Poloxamer-Botulinum Toxin Pharmaceutical Composition

A 67 year old man suffers from chronic urinary retention due toenlargement of his prostate. Upon presentation to his physician it isdetermined that the patient undergo administration ofpoloxamer-botulinum toxin pharmaceutical composition to the prostate inorder to alleviate his urinary retention and treat the benign prostatichyperplasia. About 200 units of botulinum toxin type A (BOTOX) iscombined with 4 mL of formulation 2 (20% Poloxamer 407 in 0.9% sodiumchloride) in Example 1 for transperineal injection into the bilaterallobes of the prostate (100 units per lobe).

After about 7 days, the patient reports an improvement in voiding ofurine. His physician notes that after this treatment the patient has adecrease in post voiding residual volume and bladder pressure. Thesebeneficial effects last for about 6 months and the physician notes thatthe patient's prostate has decreased in size and reports no adverseeffects. A botulinum toxin type B (such as MYOBLOC) can also beutilized, for example, from about 250 units to about 1000 units perinjection site.

A pharmaceutical composition according to the invention disclosed hereinhas many advantages, including the following:

1. the pharmaceutical composition can be prepared free of any bloodproduct, such as albumin and therefore free of any blood productinfectious element such as a prion.

2. the pharmaceutical composition has stability and high % recovery oftoxin potency comparable to or superior to that achieved with currentlyavailable pharmaceutical compositions.

3. reduced toxicity, as assessed by either intramuscular or intravenousadministration.

4. reduced antigenicity.

Various publications, patents and/or references have been cited herein,the contents of which are herein incorporated by reference in theirentireties.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of stabilizing polysaccharides and aminoacids are within the scope of the present invention.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

I claim:
 1. A method for treating a medical or cosmetic condition, themethod comprising the step of administering to a patient athermoreversible, thermoplastic, pharmaceutical composition consistingof a biologically active botulinum toxin type A present as a complexwith human serum albumin and a thermoplastic poloxamer, wherein thethermoreversible, thermoplastic, pharmaceutical composition istransitionable from a low viscosity liquid solution for easy passagethrough a needle prior to administration by injection to a patient to agel after administration.
 2. The method of claim 1, wherein thethermoplastic poloxamer is a poloxamer
 407. 3. The method of claim 1,wherein treating the medical or cosmetic condition comprises treating atleast one of glabellar lines, nasolabial lines, crows feet, marionettelines and horizontal lines of the forehead.
 4. The method of claim 1,wherein treating the medical or cosmetic condition comprises treating atleast one of overactive bladder, hyperhidrosis, benign prostatichyperplasia and a dystonia.
 5. The method of claim 1, wherein thebotulinum toxin type A is present in an amount from about 5 units toabout 2750 units.
 6. The method of claim 1, further comprising the stepof placing a cooling or heating element over an area of administrationbefore or after the administration step to warm or cool the area inorder to decrease or increase the viscosity of the administeredpharmaceutical composition, in situ, in the patient.
 7. A method fortreating a medical or cosmetic condition, the method comprising thesteps of: administering to a patient a thermo-reversible thermoplastic,pharmaceutical composition consisting a biologically active botulinumtoxin type A present as a complex with human serum albumin, and athermoreversible, thermoplastic poloxamer, wherein the poloxamerstabilizes the botulinum toxin so that the botulinum toxin retainsbiological activity upon release of the botulinum toxin from thepharmaceutical composition in vivo; and wherein the thermo-reversible,thermoplastic, pharmaceutical composition is transitionable from a lowviscosity liquid solution for easy passage through a needle prior toadministration by injection to a patient to a gel after administration.8. The method of claim 7, wherein the poloxamier is a poloxamer
 407. 9.The method of claim 7, wherein treating the medical or cosmeticcondition comprises administration to at least one of glabellar lines,nasolabial lines, crows feet, marionette lines and horizontal lines ofthe forehead.